Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.1.26.4 (RNase H)
2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Reverse transcriptase (RT) is a modular enzyme carrying polymerase and ribonuclease H (RNase H) activities in separable domains. Retroviral replication requires both of these activities. The RNase H domain is responsible for hydrolysis of the RNA portion of RNA x DNA hybrids, and this activity requires the presence of divalent cations (Mg2+ or Mn2+) that bind its active site. This domain is a part of a large family of homologous RNase H enzymes of which the RNase HI protein from Escherichia coli is the best characterized. Although the isolated RNase H domain from human immunodeficiency virus RT is inactive, the Moloney murine leukemia virus (MMLV) domain is active in the absence of the polymerase domain, making functional studies more accessible. Using circular dichroism spectroscopy, we characterized the stability and folding of two different fragments of MMLV RT that retain RNase H activity. The smaller fragment corresponding to the 157 C-terminal residues of RT is predominantly unfolded in the absence of divalent cations, but folding can be induced by the addition of metal. The larger fragment corresponding to the 175 C-terminal residues, however, is stably folded in the absence of metal. Thus, an 18 residue N-terminal extension outside the region homologous to E. coli RNase HI is important for the structural stability of the RNase H domain of MMLV RT. Therefore, this region should be considered part of the RNase H domain.
...
PMID:Folding the ribonuclease H domain of Moloney murine leukemia virus reverse transcriptase requires metal binding or a short N-terminal extension. 974 51

Two RNases H of mammalian tissues have been described: RNase HI, the activity of which was found to rise during DNA replication, and RNase HII, which may be involved in transcription. RNase HI is the major mammalian enzyme representing around 85% of the total RNase H activity in the cell. By using highly purified calf thymus RNase HI we identified the sequences of several tryptic peptides. This information enabled us to determine the sequence of the cDNA coding for the large subunit of human RNase HI. The corresponding ORF of 897 nt defines a polypeptide of relative molecular mass of 33,367, which is in agreement with the molecular mass obtained earlier by SDS/PAGE. Expression of the cloned ORF in Escherichia coli leads to a polypeptide, which is specifically recognized by an antiserum raised against calf thymus RNase HI. Interestingly, the deduced amino acid sequence of this subunit of human RNase HI displays significant homology to RNase HII from E. coli, an enzyme of unknown function and previously judged as a minor activity. This finding suggests an evolutionary link between the mammalian RNases HI and the prokaryotic RNases HII. The idea of a mammalian RNase HI large subunit being a strongly conserved protein is substantiated by the existence of homologous ORFs in the genomes of other eukaryotes and of all eubacteria and archaebacteria that have been completely sequenced.
...
PMID:Cloning of the cDNA encoding the large subunit of human RNase HI, a homologue of the prokaryotic RNase HII. 978 7

Antisense oligonucleotides (ODNs) and peptide nucleic acids (PNAs) are potential therapeutics for eradication of malignancies, viral infections, and other pathologies. However, ODNs and PNAs in general are unable to cross cellular membranes and blood-tissue barriers, such as the blood-brain barrier (BBB), which is only permeable to lipophilic molecules of molecular weight <600 Da. Cellular delivery systems based on conjugates of streptavidin (SA) and the OX26 monoclonal antibody directed to the transferrin receptor may be employed as a universal carrier for the transport of mono-biotinylated peptides, ODNs, or PNAs. 3'-Biotinylation of phosphodiester (PO)-ODN produces complete protection of ODN against serum and cellular 3'-exonucleases, facilitating the conjugation to avidin-based delivery systems and maintaining the activation of RNase H. These delivery systems markedly increased the cellular uptake and antisense efficacy of 3'-biotinylated ODNs in models of Alzheimer's disease and HIV-AIDS. In vivo brain delivery studies demonstrated that 3'-protected PO-ODNs and PO-phosphorothioate(PS)-ODN hybrids containing a single PO linkage are subjected to endonuclease degradation in vivo. On the contrary PS-ODNs, which were also protected at 3'-terminus by biotinylation, are metabolically stable in vivo and resistant to exo/endonuclease degradation. However, because of the strong binding of these oligomers to plasma protein, PS-ODNs are poorly transported into the brain through the BBB by the OX26-SA delivery vector following intravenous administration. PNAs are also resistant to exo/endonuclease and protease degradation, and these molecules biotinylated at the amino terminal group were transported into the brain by the OX26-SA delivery system with brain uptake levels comparable to that of morphine. Using the rev gene of HIV as a model target, RNase protection assays and cell-free translation arrest showed that the PNA-OX26-SA conjugate maintained active recognition and inactivation of target mRNA, respectively. The overall experimental evidence suggests that PNA-OX26-SA conjugates represent optimal antisense molecules for drug delivery to the brain.
...
PMID:Drug delivery of antisense molecules to the brain for treatment of Alzheimer's disease and cerebral AIDS. 981 82

Ribonucleases H (RNases H) comprise a family of metal-dependent enzymes that catalyze the hydrolysis of the 3'-O---P bond of RNA in RNA.DNA hybrids. The mechanism by which RNases H use active-site metal(s) for catalysis is unclear. Based upon the seemingly contradictory structural observations of one divalent metal bound to Escherichia coli RNase HI and two divalent metals bound to the HIV RNase H domain, two models explaining RNase H metal dependence have been proposed: a one-metal mechanism and a two-metal mechanism. In this paper, we show that the Mn2+-dependent activity of E. coli RNase HI is not consistent with either of these mechanisms. RNase H activity in the presence of Mn2+ is complex, with activation and inhibition of the enzyme at low and high Mn2+ concentrations, respectively. Mutations at Asp-134 result in a partial loss of this inhibition, with little effect on activation. Neutralization of His-124 by mutation to Ala results in an enzyme with a significantly decreased specific activity and an absolute loss of Mn2+ inhibition. Inhibition by high Mn2+ concentrations is shown to be due to a reduction in kcat; this attenuation has a critical dependence on the presence of His-124. Based upon these results, we propose an "activation/attenuation" model explaining the metal dependence of RNase H activity where one metal is required for enzyme activation and binding of a second metal is inhibitory.
...
PMID:Activation/attenuation model for RNase H. A one-metal mechanism with second-metal inhibition. 985 71

Database searches indicated that the genome of Bacillus subtilis contains three different genes encoding RNase H homologues. The ypdQ gene encodes an RNase HI homologue with 132 amino acid residues, whereas the rnh and ysgB genes encode RNase HII homologues with 255 and 313 amino acid residues, respectively. RNases HI and HII show no significant sequence similarity. These genes were individually expressed in Escherichia coli; the recombinant proteins were purified, and their enzymatic properties were compared with those of E. coli RNases HI and HII. We found that the ypdQ gene product showed no RNase H activity. The 2.2 kb pair genomic DNA containing this gene did not suppress the RNase H deficiency of an E. coli rnhA mutant, indicating that this gene product shows no RNase H activity in vivo as well. In contrast, the rnh (rnhB) gene product (RNase HII) showed a preference for Mn2+, as did E. coli RNase HII, whereas the ysgB (rnhC) gene product (RNase HIII) exhibited a Mg2+-dependent RNase H activity. Oligomeric substrates digested with these enzymes indicate similar recognition of these substrates by B. subtilis and E. coli RNases HII. Likewise, B. subtilis RNase HIII and E. coli RNase HI have generated similar products. These results suggest that B. subtilis RNases HII and HIII may be functionally similar to E. coli RNases HII and HI, respectively. We propose that Mn2+-dependent RNase HII is universally present in various organisms and Mg2+-dependent RNase HIII, which may have evolved from RNase HII, functions as a substitute for RNase HI.
...
PMID:Identification of the genes encoding Mn2+-dependent RNase HII and Mg2+-dependent RNase HIII from Bacillus subtilis: classification of RNases H into three families. 988

Recently we showed that the major mammalian RNase H, RNase HI, is evolutionarily related to prokaryotic RNase HII (Frank et al., FEBS-Lett. 421, 23-26, 1998), an enzyme described to be a minor activity in E. coli. As a consequence we addressed the question of whether a human RNase H exists, sharing homology with the main E. coli enzyme, RNase HI. Employing sequence analysis of expressed sequence tags, followed by specific PCR amplification of human cDNA, we cloned, sequenced and expressed a human open reading frame, coding for a 32 kDa protein. Purification of the recombinant His(6)-tagged protein from E. coli extracts using Ni(2+)-chelating chromatography and subsequent renaturation gel assay proved that it is an active RNase H. The properties of this enzyme suggest that it is identical with the human RNase HII, previously purified by one of us (Frank et al., Nucleic Acids Res. 22, 5247-5254, 1994). Studies using a green fluorescent protein-fusion construct reveal that this protein is located in the nucleus.
...
PMID:Cloning, subcellular localization and functional expression of human RNase HII. 989 7

The backbone dynamics of Escherichia coli ribonuclease H (RNase H) is studied by a recently developed off-lattice Monte Carlo/Metropolis simulation technique. A low-resolution model (virtual-bond model) is used together with knowledge-based potentials. The calculated mean-square fluctuations in alpha carbons are in good agreement with crystallographic temperature factors. The conformations generated around the native state are analyzed by time-dependent orientational and conformational correlation functions to study the internal motions of RNase at different time windows. A correlation between the free-energy changes for native-state hydrogen exchange (HX) and the extent of the autocorrelation in the rotations of the virtual bonds at long times has been observed. Cross-correlations between the rotations of the bonds, which are near-neighbor in the sequence, are effective in all time windows and help the secondary structures to preserve their kinetic stability. On the other hand, the existence of cross-correlations at long times help the tertiary contacts be maintained. The order parameter of NH bond vector for each residue has been calculated and compared with 15N-NMR relaxation measurements.
...
PMID:Characterization of internal motions of Escherichia coli ribonuclease H by Monte Carlo simulation. 1008 65

Two genes encoding functional RNase H (EC 3.1.26.4) were isolated from a gram-positive bacterium, Bacillus subtilis 168. Two DNA clones exhibiting RNase H activities both in vivo and in vitro were obtained from a B. subtilis DNA library. One (28.2 kDa) revealed high similarity to Escherichia coli RNase HII, encoded by the rnhB gene. The other (33.9 kDa) was designated rnhC and encodes B. subtilis RNase HIII. The B. subtilis genome has an rnhA homologue, the product of which has not yet shown RNase H activity. Analyses of all three B. subtilis genes revealed that rnhB and rnhC cannot be simultaneously inactivated. This observation indicated that in B. subtilis both the rnhB and rnhC products are involved in certain essential cellular processes that are different from those suggested by E. coli rnh mutation studies. Sequence conservation between the rnhB and rnhC genes implies that both originated from a single ancestral RNase H gene. The roles of bacterial RNase H may be indicated by the single rnhC homologue in the small genome of Mycoplasma species.
...
PMID:Isolation of RNase H genes that are essential for growth of Bacillus subtilis 168. 1009 89

The mitochondrial genome of Plasmodium falciparum encodes highly fragmented rRNAs. Twenty small RNAs which are putative rRNA fragments have been found and 15 of them have been identified as corresponding to specific regions of rRNA sequence. To investigate the possible interactions between the fragmented rRNAs in the ribosome, we have mapped the ends of many of the small transcripts using primer extension and RNase protection analysis. Results obtained from these studies revealed that some of the rRNA transcripts were longer than the sequences which encode them. To investigate these size discrepancies, we performed 3' RACE PCR analysis and RNase H mapping. These analyses revealed non-encoded oligo(A) tails on some but not all of these small rRNAs. The approximate length of the oligo(A) tail appears to be transcript-specific, with some rRNAs consistently showing longer oligo(A) tails than others. The oligoadenylation of the rRNAs may provide a buffer zone against 3' exonucleolytic attack, thereby preserving the encoded sequences necessary for secondary structure interactions in the ribosome.
...
PMID:The fragmented mitochondrial ribosomal RNAs of Plasmodium falciparum have short A tails. 1032 33

In addition to the conserved and well-defined RNase H domain, eukaryotic RNases HI possess either one or two copies of a small N-terminal domain. The solution structure of one of the N-terminal domains from Saccharomyces cerevisiae RNase HI, determined using NMR spectroscopy, is presented. The 46 residue motif comprises a three-stranded antiparallel beta-sheet and two short alpha-helices which pack onto opposite faces of the beta-sheet. Conserved residues involved in packing the alpha-helices onto the beta-sheet form the hydrophobic core of the domain. Three highly conserved and solvent exposed residues are implicated in RNA binding, W22, K38 and K39. The beta-beta-alpha-beta-alpha topology of the domain differs from the structures of known RNA binding domains such as the double-stranded RNA binding domain (dsRBD), the hnRNP K homology (KH) domain and the RNP motif. However, structural similarities exist between this domain and the N-terminal domain of ribosomal protein L9 which binds to 23 S ribosomal RNA.
...
PMID:NMR structure of the N-terminal domain of Saccharomyces cerevisiae RNase HI reveals a fold with a strong resemblance to the N-terminal domain of ribosomal protein L9. 1044 44


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---